Compositions, methods and uses for the treatment of diabetic neuropathies

ABSTRACT

The present invention relates to compositions and methods for the treatment of diabetic neuropathies. More particularly, this invention relates to compositions and methods for promoting nerve regeneration in a subject suffering from, or at risk of suffering from a diabetic neuropathy, and for protecting nervous system cells against the stresses resulting from the perturbation of glucose homeostasis. More specifically, the present invention relates to novel single or combination therapies for diabetic neuropathies, comprising diabetic peripheral neuropathy, diabetic proximal neuropathy, diabetic autonomic neuropathy and diabetic focal neuropathy, based on restoring dorsal root ganglion neurons and other nerves integrity.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treating diabetic neuropathies in a subject in need thereof. More specifically, the present invention relates to novel therapies, in particular, combinatorial therapies of diabetic neuropathies.

BACKGROUND OF THE INVENTION

Neuropathy is the most common complication affecting diabetic patients, approximately half of them being concerned. The number of diabetic patients, an estimated 381 million people in 2013, is expected to double by 2030. Already today, the diabetic neuropathies can be considered as one of the major public health concerns and can be defined as an unmet medical need: efficacy of existing medications for treating diabetic neuropathies—even symptomatic—is limited, and none of these medications can be unambiguously classified as a disease-modifying intervention.

Neuropathy is a common complication of both type I and type II diabetes. The vast majority of diabetic patients develop a distal symmetric polyneuropathy which can be defined as a sensory peripheral neuropathy and manifested in a distal axonopathy. Both myelinated and unmyelinated neurons are involved. Diabetic neuropathies mainly affect neurons with the longest axons, leading to pathophysiological symptoms in the feet and hands. Later, these symptoms may progress proximally to the arms and legs. Some patients can develop autonomic and cranial forms of diabetic neuropathies which could be associated with myocardial infarction, malignant arrhythmia and sudden death. Symptoms associated with peripheral neuropathy include pain, the principal source of disability, as well as tingling, weakness and balance difficulties. Importantly, loss of sensation (numbness) is also frequently associated with diabetic neuropathies and may lead to the development of small infections and ulcerations sometimes requiring amputation.

The cellular mechanisms involved in the pathogenesis of diabetic neuropathies are poorly understood. It is thought to result from hyperglycaemia-induced injuries associated with insulin resistance, due to excessive production of potentially toxic intermediate metabolic substances such as sorbitol or modifications of cellular proteins leading to oxidative and inflammatory stress. Peripheral sensory neurons and endothelial cells from small blood vessels supplying peripheral nerves are supposed to be particularly vulnerable to these pathological factors [1,2]. In addition to hyperglycemic stress, epidemiological studies have shown that dyslipidemia is also strongly associated with development of microvascular complications in diabetic patients and, thereby etiologically contributes to progression of neuropathic complications in affected individuals.

As mentioned above, pain is the most severe and constant manifestation that disables the quality of life of diabetic patients with peripheral neuropathy. Respectively,—from medical point of view,—alleviation of pain is considered as one of the main goals of therapeutics targeting diabetic neuropathies. A few medications are used now and several are being developed for symptomatic treatment of peripheral diabetic neuropathies. It was reported that more than 50% reduction in pain can be achieved in only one-third of patients despite use of available therapy. Mostly, drugs alleviating painful symptoms are exemplified by anticonvulsant and antidepressant drugs such as pregabalin or duloxetine, among the most preferred ones [3-5]. Use of the symptomatic drugs is still under debate due to issues about their efficacy, and safety concerns such as dependency, tolerance and addiction.

Some drugs like lipoic acid that is a standard of care in Germany, benfotiamine, actovegin or aldose reductase inhibitors are considered as putative disease-modifying interventions, but additional robust clinical evidences are required to prove their efficacy and absence of significant adverse effects [6-8].

Therapeutic options for treating diabetic neuropathies may be either symptomatic or disease-modifying. Though of enormous therapeutic importance, treatment of symptoms does not necessarily prevent the progression of the disease. Therefore, there is an urgent medical need for developing efficient, safe, symptomatic and disease-modifying drugs that attenuate, slow or stop progression or even reverse peripheral neuropathy and improve quality of life in most diabetic patients suffering from this debilitating complication.

SUMMARY OF INVENTION

The purpose of the present invention is to provide novel and efficient treatments for diabetic neuropathies. More particularly, the present invention proposes new compositions based on single drugs or drug combinations for use in treating or preventing a diabetic neuropathy in subjects suffering from diabetes such as diabetes type I or II. Typically, the compositions and methods provided by the inventors may be used for treating a subject suffering from, at risk of suffering from, or likely to suffer from a diabetic neuropathy. The diabetic neuropathy treated according to the invention may be a diabetic peripheral neuropathy, a diabetic proximal neuropathy, a diabetic autonomic neuropathy or a diabetic focal neuropathy. Advantageously, the compositions of the invention are used to promote nerve regeneration in subjects suffering from or at risk of suffering from a diabetic neuropathy.

The compositions of the invention are also used to efficiently protect nervous system cells against the stresses resulting from the perturbation of glucose homeostasis, in subjects suffering from diabetes type I or II, pre-diabetes, chronic hyperglycaemia, impaired fasting glycaemia, impaired glucose tolerance or obesity.

An object of this invention therefore relates to compositions for use in the curative or preventive treatment of a diabetic neuropathy in a subject in need thereof, comprising at least two drugs selected from acamprosate, almitrine, amlexanox, azelastine, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene.

Preferably, the composition of the invention comprises at least two drugs selected from acamprosate, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, ifenprodil, levosimendan, mexiletine, nicergoline, rilmenidine, tolperisone, torasemide and triamterene.

Another object of the invention relates to compositions for the treatment of a diabetic neuropathy, comprising:

-   -   at least one drug selected from acamprosate, baclofen,         carbetapentane, cimetidine, cinacalcet, dexbrompheniramine,         diethylcarbamazine, ifenprodil, levosimendan, mexiletine,         nicergoline, rilmenidine, tolperisone, torasemide and         triamterene, and     -   at least one distinct drug selected form acamprosate, almitrine,         amlexanox, azelastine, baclofen, carbetapentane, cimetidine,         cinacalcet, dexbrompheniramine, diethylcarbamazine,         diprophylline, D-mannose, fenspiride, fexofenadine, idebenone,         ifenprodil, levosimendan, mexiletine, nicergoline, piribedil,         rilmenidine, tolfenamic acid, tolperisone, torasemide and         triamterene.

A further object of the invention relates to compositions for treating a diabetic neuropathy, comprising:

-   -   at least one drug selected from acamprosate, cinacalcet,         levosimendan, torasemide and triamterene, and     -   at least one distinct drug selected form acamprosate, almitrine,         amlexanox, azelastine, baclofen, carbetapentane, cimetidine,         cinacalcet, dexbrompheniramine, diethylcarbamazine,         diprophylline, D-mannose, fenspiride, fexofenadine, idebenone,         ifenprodil, levosimendan, mexiletine, nicergoline, piribedil,         rilmenidine, tolfenamic acid, tolperisone, torasemide and         triamterene.

A further object of the invention relates to composition for the treatment of a diabetic neuropathy, comprising:

-   -   at least one drug selected from acamprosate, cinacalcet,         levosimendan, torasemide and triamterene, and     -   at least one distinct drug selected form acamprosate, baclofen,         carbetapentane, cimetidine, cinacalcet, dexbrompheniramine,         diethylcarbamazine, ifenprodil, levosimendan, mexiletine,         nicergoline, rilmenidine, tolperisone, torasemide and         triamterene.

A particular object of the invention relates to compositions for use in the treatment of a diabetic neuropathy, comprising at least one of the following drug combinations:

-   -   acamprosate and cinacalcet,     -   acamprosate and levosimendan,     -   acamprosate and torasemide,     -   acamprosate and triamterene,     -   acamprosate and baclofen,     -   cinacalcet and levosimendan,     -   cinacalcet and torasemide,     -   cinacalcet and triamterene,     -   torasemide and triamterene,     -   levosimendan and torasemide, or     -   levosimendan and triamterene.

The compositions of the invention may further comprise one or more additional active compounds. For example, at least one drug selected from duloxetine and pregabalin may be added to the compositions of the invention.

Alternatively, the compositions of the invention may further comprise at least one antidiabetic agent selected from acarbose, acetohexamide, alogliptin, berberine, bezaflbrate, bromocriptine, buformin, carbutamide, chlorpropamide, chromium picolinate, ciprofibrate, do fibrate, colesevelam, dexfenfluramine, dutogliptin, exenatide, fenofibrate, gemfibrozil, gemigliptin, glibenclamide, glibornuride, glicetanile, gliclazide, glimepiride, glipizide, gliquidone, glisentide, glyclopyramide, imidapril, insulin, inulin, lipoic acid, linagliptin, liraglutide, mecobalamin, metformin, miglitol, mitiglinide, nateglinide, orlistat, phenformin, pioglitazone, pramlintide, repaglinide, rosiglitazone, saxagliptin, sitagliptin, tolazamide, tolbutamide, vildagliptin or voglibose. Preferably, the antidiabetic agent used in the compositions of the invention, is metformin.

Also, the drugs as used in the present invention may be in the form of salts, hydrates, esters, ethers, acids, amides, racemates or isomers. They may also be in the form of sustained-release formulations, prodrugs or derivatives of the drugs. Preferably, the drug is used as such or in the form a salt, hydrate, ester, ether or sustained release form thereof. In another preferred embodiment, a prodrug or derivative is used.

The compositions of the invention typically comprise one or several pharmaceutically acceptable excipients or carriers.

As it will be further disclosed in the present application, the drugs in a combinatorial therapy according to the invention may be administered simultaneously, separately, sequentially and/or repeatedly to the subject.

Another object of this invention relates to a method for treating a diabetic neuropathy in a mammalian subject in need thereof, preferably a human subject, the method comprising administering to said subject an effective amount of a combination of the invention.

The invention may be used for treating a diabetic neuropathy in any mammalian subject, preferably in any human subject, at any stage of the disease. Furthermore, the invention may be used in a subject suffering from, at risk of suffering from, or likely to suffer from a diabetic neuropathy. As it will be disclosed in the examples, the compositions of the invention are able to ameliorate the pathological condition of said subjects.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new therapeutic approaches for treating diabetic neuropathies. In particular, the invention provides novel drug combinations and methods, which are effective in protecting nervous tissues against the stresses resulting from the perturbation of glucose homeostasis, or in restoring said tissues in a diabetic subject, and which may be used in any mammalian subject suffering from diabetes.

The invention therefore relates to compositions and methods for the treatment of diabetic neuropathies.

DEFINITIONS

Within the context of the invention, the term “treatment” designates a prophylactic, preventive or curative treatment.

Consequently such treatment can be used in a subject suffering from, at risk of suffering from, or likely to suffer from diabetic neuropathies. More particularly, such a subject can be diagnosed as suffering from, or at risk of suffering from, diabetes type I or II, pre-diabetes, chronic hyperglycaemia, impaired fasting glycaemia, impaired glucose tolerance or obesity.

The term “treatment” also includes the correction, the retardation of the onset, or the retardation of the progression of the disease. The term “treatment” also includes the correction, the retardation or the reduction of symptoms of the disease. Consequently, the term treatment includes the prevention, retardation or reduction or improvement of any motor, autonomic and/or sensory impairment caused by diabetic neuropathies.

“Motor symptoms” include uncontrollable contractions, fasciculation weakness, or complete unresponsiveness of muscle(s) innervated by injured nerves.

“Autonomic symptoms” refers to impairments from neuronal origin of cardiovascular, sudomotor, thermoregulatory, urinary, gastrointestinal, and/or reproductive systems.

“Sensory symptoms” include pain, numbness, or the loss of sensation in the areas innervated by the injured nerves.

Within the context of the invention the term “diabetic neuropathies” or “a diabetic neuropathy” refers herein to the different types of diabetic neuropathies including diabetic peripheral neuropathy, diabetic proximal neuropathy, diabetic autonomic neuropathy (as for example diabetic retinal neuropathy) and diabetic focal neuropathy.

More particularly, the term “treatment” encompasses the partial or total recovery of dorsal root ganglion (DRG) neurons or other nerves from damages by hyperglycaemia, oxidative stress, polyol pathway over-activity, advanced glycation end products and/or hypoxia/ischemia resulting from diabetes. Accordingly, the treatment also refers to nerve regeneration through re-myelination processes, axonal growth, enhancement of neuronal growth, and/or restoration of either morphological or functional features of the nerves.

In the context of the invention, the term “treatment” also designates the improvement of small blood vessels which supply nerves and which are damaged by high blood glucose.

The term “combination” or “combinatorial therapy” or “combinatory treatment” designates a treatment wherein at least two compounds are co-administered to a subject to cause a biological effect. In a combined therapy according to this invention, the at least two drugs may be administered together or separately, at the same time or sequentially. Simultaneous administration is not required, as long as the drugs produce a combined or synergistic effect in the organism to improve the patient conditions. Also, the at least two drugs may be administered through different routes and protocols. As a result, although they may be formulated together, the drugs of a combination may also be formulated separately.

Within the context of the invention, the term “compound” or “drug” as identified by its name or CAS number is meant to designate the chemical compound as specifically named or identified with its corresponding CAS number, as well as any pharmaceutically acceptable salt, hydrate, isomer, racemate, conjugate or derivative thereof, of any chemical purity.

The term “derivative” includes any functionally and structurally related compound, such as an acid, amide, ester, ether, acetylated, hydroxylated or an alkylated (C1-C6) variant of such a compound. The term “derivative” also includes structurally related compound having lost one or more substituent as listed above. The term “derivative” particularly includes prodrugs and metabolites of said compound. Preferred derivatives of a compound are molecules having a substantial degree of similarity to said compound, as determined by known methods. Similar compounds along with their index of similarity to a parent molecule can be found in numerous databases such as PubChem (http://pubchem.ncbi.nlm.nih.gov/search/) or DrugBank (http://www.drugbank.ca/). In a more preferred embodiment, derivatives should have a Tanimoto similarity index greater than 0.4, preferably greater than 0.5, more preferably greater than 0.6, even more preferably greater than 0.7 with a parent drug. The Tanimoto similarity index is widely used to measure the degree of structural similarity between two molecules. Tanimoto similarity index can be computed by software such as the Small Molecule Subgraph Detector [9,10] available online (http://www.ebi.ac.uk/thornton-srv/software/SMSD/). As mentioned above, a derivative should be both structurally and functionally related to a parent compound, preferably, in vitro, it should protect nerve cells from stresses resulting from a high glucose condition. More preferably it should retain at least a part of the activity of the parent drug, more preferably it should promote in vitro the outgrowth of neurites from DRG of spinal cord explants from streptozotocin (STZ) treated rats, or protect DRG explants from wild type animals cultured in high glucose conditions.

The term “derivative” also includes metabolites of a drug, e.g., a molecule which results from the (biochemical) modification(s) or processing of said drug after administration to an organism, usually through specialized enzymatic systems, and which displays or retains a biological activity of the drug. Metabolites have been disclosed as being responsible for much of the therapeutic action of the parent drug. In a specific embodiment, a metabolite as used herein designates a modified or processed drug that, in vitro, protects nerve cells from stresses resulting from a high glucose condition. More preferably it should retain at least part of the activity of the parent drug, preferably that promotes in vitro neurites the outgrowth of neurites from DRG of spinal cord explants from STZ-treated rats, or protect DRG explants from wild type animals cultured in high glucose conditions.

The term “prodrug” as used herein refers to any derivative (or precursor) of a compound which, when administered to a biological system (e.g., a human organism), generates said compound as a result of e.g., spontaneous chemical reaction(s), enzyme catalyzed chemical reaction(s), and/or metabolic chemical reaction(s). Prodrugs typically have the structure X-drug, wherein X is an inert carrier moiety and drug is the active compound. Usually, the prodrug is devoid of activity or less active than the drug and the drug is released from the carrier in vivo. Prodrugs can be used, for example, to improve the physicochemical properties of the drug, to target the drug to a specific tissue, to improve the pharmacokinetic and pharmacodynamic properties of the drug and/or to reduce undesirable side effects. Some of the common functional groups that are amenable to prodrug design include, but are not limited to carboxylic, hydroxyl, amine, phosphate/phosphonate and carbonyl groups. Prodrugs typically produced via the modification with these groups include, but are not limited to, esters, carbonates, carbamates, amides and phosphates. Specific technical guidance for the selection of suitable prodrugs is general common knowledge [11-15]. Furthermore, the preparation of prodrugs may be performed by conventional methods known by those skilled in the art. Methods which can be used to synthesize prodrugs are described in numerous reviews on the subject [11-18].

The term “metabolite” of a drug as used herein refers to a molecule which results from the (biochemical) modification(s) or processing of said drug after administration to an organism, usually through specialized enzymatic systems, and which displays or retains a biological activity of the drug. Metabolites have been disclosed as being responsible for much of the therapeutic action of the parent drug.

The term “salt” refers to a pharmaceutically acceptable and relatively non-toxic, inorganic or organic acid or basic addition salt of a compound of the present invention. Pharmaceutical salt formation typically consists in pairing an acidic, basic or zwitterionic drug molecule with a counter ion to create a salt version of the drug. A wide variety of chemical species can be used in neutralization reaction. Though most of salts of a given active principle are bioequivalents, some may have, among others, increased solubility or bioavailability properties. Salt selection is now a common standard operation in the process of drug development as taught by H. Stahl and C. G Wermuth in their handbook [19].

In a preferred embodiment, the designation of a compound is meant to designate the compound per se, as well as any pharmaceutically acceptable salt, hydrate, isomer, or racemate thereof.

In a more preferred embodiment, the designation of a compound is meant to designate the compound as specifically designated per se, as well as any pharmaceutically acceptable salt thereof.

In a particular embodiment, a sustained-release formulation of the compound is used.

Compositions and Methods for Treating Diabetic Neuropathies

By a comprehensive integration of experimental data covering results of cell biology studies, expression profiling experiments and genetic association studies, the inventors have been able to select drugs which, alone and/or in combination(s), effectively alter relevant pathways for the prevention or control of the pathogenesis of diabetic neuropathies and thereby represent new therapeutic approaches for treating diabetic neuropathies. These drugs used alone or in combination are efficient in promoting nerve regeneration including re-myelination processes of nerves, axonal growth, enhancement of neuronal growth, and/or restoration of either morphological or functional features of the nerves.

As shown in the experimental section, these drugs and combinations thereof are efficient in protecting DRG from degeneration or death induced by hyperglycaemia and/or promoting neurite outgrowth in vitro in diabetes related conditions, and/or correcting diabetes related neuropathic symptoms in vivo. Consequently, they represent new therapeutic approaches for the treatment of diabetic neuropathies in a subject in need thereof.

In this regard, in a specific embodiment, this invention relates to compositions comprising at least one compound selected from the group consisting of acamprosate, almitrine, amlexanox, azelastine, baclo fen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergo line, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof, for use in the treatment of diabetic neuropathies in a subject in need thereof.

The invention also relates to the use of at least one compound as listed above in the manufacture of a medicament for treating diabetic neuropathies in a subject in need thereof.

The invention also relates to a method for treating diabetic neuropathies in a subject in need thereof, comprising administering to the subject at least one compound as listed above. Illustrative CAS numbers for each of the selected compounds are provided in table 1 below:

TABLE 1 Drug Name CAS number acamprosate 77337-76-9; 77337-73-6 almitrine 27469-53-0; 29608-49-9 amlexanox 68302-57-8; azelastine 58581-89-8; 79307-93-0 baclofen 1134-47-0; 66514-99-6; 69308-37-8; 70206-22-3; 63701-56-4; 63701-55-3 carbetapentane 77-23-6; 23142-01-0; 1045-21-2 cimetidine 51481-61-9; 70059-30-2 cinacalcet 226256-56-0; 364782-34-3 dexbrompheniramine 86-22-6; 980-71-2; 2391-03-9 diethylcarbamazine 90-89-1; 1642-54-2 diprophylline 479-18-5 D-mannose 3458-28-4 fenspiride 5053-06-5; 5053-08-7 fexofenadine 83799-24-0; 138452-21-8; 153439-40-8; 139965-10-9; 139965-11-0 idebenone 58186-27-9 ifenprodil 23210-56-2; 23210-58-4 levosimendan 141505-33-1 mexiletine 5370-01-4; 31828-71-4 nicergoline 27848-84-6 piribedil 3605-01-4 rilmenidine 54187-04-1; 85409-38-7 tolfenamic acid 13710-19-5 tolperisone 728-88-1 torasemide 56211-40-6; 72810-59-4 triamterene 1226-52-4; 396-01-0

As mentioned in the examples, compositions of the invention comprising the above compounds, either individually or in combination, are efficient in protecting DRG and neurite outgrowth from high glucose concentration toxicity. Moreover in vivo, compositions of the invention are efficient in improving neuropathies experienced by diabetic subjects.

Furthermore, the inventors have found that acamprosate, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, ifenprodil, levosimendan, mexiletine, nicergoline, rilmenidine, tolperisone, torasemide and triamterene are particularly efficient in protecting DRG from apoptosis and/or in improving neurite outgrowth.

In this regard, a preferred embodiment of the invention relates to a composition for use in the treatment of diabetic neuropathies, the composition comprising at least one compound selected from acamprosate, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, ifenprodil, levosimendan, mexiletine, nicergoline, rilmenidine, tolperisone, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof.

The above molecules may be used alone or, preferably, in combination therapies to provide the most efficient clinical benefit. Drug combinations are particularly advantageous because they can affect different pathways and thus are more effective. Also, because of their efficacy and mode of action, the drug combinations can be used at low dosages, which is a further very substantial advantage. Also, more preferably, drug compositions of the invention may comprise 1, 2, 3, 4 or 5 distinct drugs, even more preferably 2, 3 or 4 distinct drugs for combinatorial treatment.

In this regard, an object of this invention relates to compositions comprising a combination of at least two compounds chosen from the group consisting of from acamprosate, almitrine, amlexanox, azelastine, baclo fen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or salts, prodrugs, derivatives, or sustained release formulations thereof, for use in the treatment of diabetic neuropathies in a subject in need thereof.

A preferred object of this invention relates to compositions comprising a combination of at least two compounds selected from the group consisting of acamprosate, baclo fen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, ifenprodil, levosimendan, mexiletine, nicergo line, rilmenidine, tolperisone, torasemide and triamterene, or salts, prodrugs, derivatives, or sustained release formulations thereof, for use in the treatment of diabetic neuropathies in a subject in need thereof.

Another preferred object of this invention relates to a composition comprising:

-   -   at least one drug selected from acamprosate, baclofen,         carbetapentane, cimetidine, cinacalcet, dexbrompheniramine,         diethylcarbamazine, ifenprodil, levosimendan, mexiletine,         nicergoline, rilmenidine, tolperisone, torasemide and         triamterene, or a salt, prodrug, derivative, or sustained         release formulation thereof, and     -   at least one distinct drug selected form acamprosate, almitrine,         amlexanox, azelastine, baclo fen, carbetapentane, cimetidine,         cinacalcet, dexbrompheniramine, diethylcarbamazine,         diprophylline, D-mannose, fenspiride, fexofenadine, idebenone,         ifenprodil, levosimendan, mexiletine, nicergoline, piribedil,         rilmenidine, tolfenamic acid, tolperisone, torasemide and         triamterene, or a salt, prodrug, derivative, or sustained         release formulation thereof, for use in the treatment of         diabetic neuropathies in a subject in need thereof.

A particularly preferred object of this invention relates to a composition comprising acamprosate or a salt, prodrug, derivative, or sustained release formulation thereof in a combination with at least one drug selected from almitrine, amlexanox, azelastine, baclo fen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof, for use in the treatment of diabetic neuropathies in a subject in need thereof.

Another preferred object of this invention relates to a composition comprising levosimendan or a salt, prodrug, derivative, or sustained release formulation thereof in a combination with at least one drug selected from acamprosate, almitrine, amlexanox, azelastine, baclo fen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or pharmaceutically acceptable salts thereof, for use in the treatment of diabetic neuropathies in a subject in need thereof.

Another particularly preferred object of this invention relates to a composition comprising cinacalcet or a salt, prodrug, derivative, or sustained release formulation thereof in a combination with at least one drug selected from acamprosate, almitrine, amlexanox, azelastine, baclo fen, carbetapentane, cimetidine, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or pharmaceutically acceptable salts thereof for use in the treatment of diabetic neuropathies in a subject in need thereof.

A particular object of this invention relates to a composition comprising torasemide or a salt, prodrug, derivative, or sustained release formulation thereof in a combination with at least one drug selected from acamprosate, almitrine, amlexanox, azelastine, baclo fen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof, for use in the treatment of diabetic neuropathies in a subject in need thereof.

Another preferred object of this invention relates to a composition comprising triamterene or a salt, prodrug, derivative, or sustained release formulation thereof in a combination with at least one drug selected from acamprosate, almitrine, amlexanox, azelastine, baclo fen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone and torasemide, or a salt, prodrug, derivative, or sustained release formulation thereof, for use in the treatment of diabetic neuropathies in a subject in need thereof.

In an even more preferred embodiment, compositions of this invention comprise at least one of the following drug combinations, for combined, separate or sequential administration:

-   -   acamprosate and cinacalcet,     -   acamprosate and levosimendan,     -   acamprosate and torasemide,     -   acamprosate and triamterene,     -   acamprosate and baclofen,     -   cinacalcet and levosimendan,     -   cinacalcet and torasemide,     -   cinacalcet and triamterene,     -   torasemide and triamterene,     -   levosimendan and torasemide, or     -   levosimendan and triamterene, or         salts, prodrugs, derivatives, or sustained release formulation         for use in the treatment of diabetic neuropathies in a subject         in need thereof.

Another object of this invention resides in the use of a composition as defined above for promoting nerve regeneration, i.e. promoting processes of nerve re-myelination, axonal growth, neuronal growth, and/or of restoration of either morphological or functional features of the nerves in a diabetic subject in need thereof.

A further object of this invention resides in the use of any of the compositions as defined above for the manufacture of a medicament for the treatment of diabetic neuropathies in a subject in need thereof.

Another object of this invention relates to the use of any of the compositions as defined above for the manufacture of a medicament for promoting nerve regeneration, i.e. promoting processes of nerve re-myelination, axonal growth, enhancement of neuronal growth, and/or of restoration of either morphological or functional features of the nerves in a diabetic subject in need thereof.

As indicated previously, in a composition or combination therapy of this invention, the compounds or drugs may be formulated together or separately, and administered together, separately or sequentially.

Consequently, an object of the invention is a method of treating diabetic neuropathies, the method comprising sequentially administering to a subject in need thereof an effective amount of a drug as defined above.

Another object of the invention is also a method of treating diabetic neuropathies, the method comprising simultaneously, separately or sequentially administering to a subject in need thereof an effective amount of drugs combined as defined above.

Consequently, in an embodiment, the invention relates to a method of treating diabetic neuropathies in a subject in need thereof, comprising administering to the subject an effective amount of at least one drug selected from acamprosate, almitrine, amlexanox, azelastine, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergo line, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof.

In a preferred embodiment, the invention relates to a method of treating diabetic neuropathies in a subject in need thereof, comprising administering simultaneously, separately or sequentially to the subject an effective amount of at least two drugs selected from acamprosate, almitrine, amlexanox, azelastine, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergo line, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or salts, prodrugs, derivatives, or sustained release formulations thereof.

In a preferred embodiment, the invention relates to a method of treating diabetic neuropathies in a subject in need thereof, comprising administering simultaneously, separately or sequentially to the subject an effective amount of at least two drugs selected from acamprosate, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, ifenprodil, levosimendan, mexiletine, nicergoline, rilmenidine, tolperisone, torasemide and triamterene, or salts, prodrugs, derivatives, or sustained release formulations thereof.

In another preferred embodiment, this invention relates to a method of treating diabetic neuropathies in a subject in need thereof, comprising administering simultaneously, separately or sequentially to the subject an effective amount a composition comprising acamprosate or a salt, prodrug, derivative, or sustained release formulation thereof, with at least one drug selected from almitrine, amlexanox, azelastine, baclo fen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof.

In another preferred embodiment, this invention relates to a method of treating diabetic neuropathies in a subject in need thereof, comprising administering simultaneously, separately or sequentially to the subject an effective amount of a composition comprising levosimendan or a salt, prodrug, derivative, or sustained release formulation, with at least one drug selected from acamprosate, almitrine, amlexanox, azelastine, baclo fen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof.

In a further embodiment, this invention relates to a method of treating diabetic neuropathies in a subject in need thereof, comprising administering simultaneously, separately or sequentially to the subject an effective amount of a composition comprising cinacalcet or a salt, prodrug, derivative, or sustained release formulation thereof, with at least one drug selected from acamprosate, almitrine, amlexanox, azelastine, baclo fen, carbetapentane, cimetidine, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof.

In another embodiment, this invention relates to a method of treating diabetic neuropathies in a subject in need thereof, comprising administering simultaneously, separately or sequentially to the subject an effective amount of a composition comprising torasemide or a salt, prodrug, derivative, or sustained release formulation thereof, with at least one drug selected from acamprosate, almitrine, amlexanox, azelastine, baclo fen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof.

In another preferred embodiment, this invention relates to a method of treating diabetic neuropathies in a subject in need thereof, comprising administering simultaneously, separately or sequentially to the subject an effective amount of a composition comprising triamterene or a salt, prodrug, derivative, or sustained release formulation thereof in a combination with at least one drug selected from acamprosate, almitrine, amlexanox, azelastine, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergo line, piribedil, rilmenidine, tolfenamic acid, tolperisone, and torasemide, or a salt, prodrug, derivative, or sustained release formulation thereof.

In a more preferred embodiment, the invention relates to a method of treating diabetic neuropathies in a subject in need thereof, comprising administering simultaneously, separately or sequentially to the subject an effective amount of at least one of the following drug combinations:

-   -   acamprosate and cinacalcet,     -   acamprosate and levosimendan,     -   acamprosate and torasemide,     -   acamprosate and triamterene,     -   acamprosate and baclofen,     -   cinacalcet and levosimendan,     -   cinacalcet and torasemide,     -   cinacalcet and triamterene,     -   torasemide and triamterene,     -   levosimendan and torasemide, or     -   levosimendan and triamterene, or         salts, prodrugs, derivatives, or sustained release formulations         thereof.

The compositions of the invention typically comprise one or several pharmaceutically acceptable carriers or excipients. Also, for use according to the present invention, the drugs or compounds are usually mixed with pharmaceutically acceptable excipients or carriers.

In this regard, a further object of this invention is a method of preparing a pharmaceutical composition, the method comprising mixing the above compounds in appropriate excipient(s) or carrier(s).

According to preferred embodiments of the invention, as indicated above, the compounds are used as such or in the form of a pharmaceutically acceptable salt, prodrug, metabolite, or sustained release formulation thereof.

Although very effective in vitro and in vivo, depending on the subject or specific condition, the above methods, compositions or combination therapies may further be used in conjunction or association or combination with additional drugs or treatments known or being developed to improve diabetic neuropathies or symptoms thereof. For instance, combinations of the invention can be used in conjunction with acetyl-L-carnitine, actovegin, allopurinol and alpha-lipoic acid and nicotinamide, alpha-lipoic acid, benfotiamine, BK-C-0701, botulinum toxin type A, nabiximols, capsaicin, carbamazepine, cilostazol, clonidine, desvenlafaxine succinate, dextromethorphan hydrobromide and quinidine sulfate, donepezil, duloxetine, epalrestat, lacosamide, eslicarbazepine, F0434, fentanyl, gabapentin, gabapentin and thiamine and cyanocobalamin, goshajinkigan, isosorbide-5-mononitrate, JWHGWT, ketamine, ketamine and clonidine, L-arginine, lidocaine, lidocaine and gabapentin, lidocaine and prilocaine, L-methylfolate and methyl cobalamine and pyridoxal 5′-phosphate, methyl cobalamine, memantine, mexiletine, morphine and naltrexone, nabilone, Neuragen®, vitamin B3, omega-3-acid ethyl esters, oxycodone, pregabalin, ranirestat, ruboxistaurin mesylate, sildenafil, sorbinil, tapentadol, tocotrienol, trafermin, tramadol and acetaminophen, tramadol and duloxetine, tramadol and pregabalin, or tramadol and acetaminophen. Illustrative CAS numbers for each of compounds listed above are provided in table 2 below:

TABLE 2 Drug Name CAS number acetyl-L-carnitine 3040-38-8; 5080-50-2 actovegin 63748-11-8 allopurinol 315-30-0; 17795-21-0 alpha-lipoic acid 1200-22-2; 1077-27-6; 1077-28-7; 62-46-4; 2319-84-8 benfotiamine 22457-89-2 botulinum toxin type A 93384-43-1 capsaicin 404-86-4 carbamazepine 298-46-4 cilostazol 73963-72-1 clonidine 4205-90-7 cyanocobalamin 68-19-9 desvenlafaxine succinate 386750-22-7 dextromethorphan 125-69-9; 6700-34-1 hydrobromide donepezil 120014-06-4; 142057-79-2; 120011-70-3; 142057-77-0 duloxetine 116539-59-4; 116539-60-7; 116817-13-1; 136434-34-9; 910138-96-4 epalrestat 82159-09-9 eslicarbazepine 104746-04-5 F0434 NA fentanyl 437-38-7; 1443-54-5 gabapentin 60142-96-3; 478296-72-9 goshajinkigan NA isosorbide-5-mononitrate 16051-77-7 JWHGWT NA ketamine 6740-88-1; 1867-66-9; 33795-24-3; lacosamide 175481-36-4 L-arginine 74-79-3 lidocaine 137-58-6; 73-78-9; L-methylfolate 134-35-0; 151533-22-1 memantine 19982-08-2 methylcobalamin 13422-55-4 mexiletine 5370-01-4; 31828-71-4 morphine 57-27-2; 64-31-3; 52-26-6 nabilone 51022-71-0 nabiximols 56575-23-6 naltrexone 16590-41-3 Neuragen ® NA nicotinamide 98-92-0 omega-3-acid ethyl esters 86227-47-6 oxycodone 76-42-6 pregabalin 148553-50-8 prilocaine 721-50-6 pyridoxal 5′-phosphate 54-47-7 quinidine sulfate 56-54-2 ranirestat 147254-64-6 ruboxistaurin mesylate 192050-59-2 sildenafil 139755-83-2 sorbinil 68367-52-2 tapentadol 175591-23-8 thiamine 59-43-8 tocotrienol 6829-55-6 trafermin 131094-16-1 tramadol 27203-92-5 vitamin B3 59-67-6 NA: not applicable (e.g., mixes, traditional medicines . . .)

In this regard, a particularly preferred embodiment relates to a combination of any of the compositions of the invention with duloxetine and/or pregabalin or a salt, prodrug, derivative, or sustained release, for use in the treatment of diabetic neuropathies in a subject in need thereof.

In a more preferred embodiment, the invention relates to a composition comprising pregabalin or pharmaceutically acceptable salts thereof and at least one drug selected from acamprosate, almitrine, amlexanox, azelastine, baclo fen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or pharmaceutically acceptable salts thereof.

In another preferred embodiment, the invention relates to a composition comprising duloxetine or pharmaceutically acceptable salts thereof and at least one drug selected from acamprosate, almitrine, amlexanox, azelastine, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or pharmaceutically acceptable salts thereof.

The above combinations comprising one or more drugs of the invention and a known drug or treatment listed in table 2, or a combination thereof, allow a diminution of the dosage of these drugs for the treatment of diabetic neuropathies.

Diabetic neuropathies being a complication of diabetes, the above mentioned drug(s) or drug combination(s) according to the present invention, may be administered to a patient undergoing a treatment for controlling glucose homeostasis. Preferably, said treatment for controlling glucose homeostasis may comprise one or more drug(s) selected from acarbose, acetohexamide, alogliptin, berberine, bezafibrate, bromocriptine, buformin, carbutamide, chlorpropamide, chromium picolinate, ciprofibrate, do fibrate, colesevelam, dexfenfluramine, dutogliptin, exenatide, fenofibrate, gemfibrozil, gemigliptin, glibenclamide, glibornuride, glicetanile, gliclazide, glimepiride, glipizide, gliquidone, glisentide, glyclopyramide, imidapril, insulin, inulin, lipoic acid, linagliptin, liraglutide, mecobalamin, metformin, miglitol, mitiglinide, nateglinide, orlistat, phenformin, pioglitazone, pramlintide, repaglinide, rosiglitazone, saxagliptin, sitagliptin, tolazamide, tolbutamide, vildagliptin and voglibose.

Illustrative CAS numbers for each of these compounds are provided in table 3 below:

TABLE 3 Drug Name CAS number Analogs of amylin pramlintide 196078-30-5 Glucagon-like peptide 1 receptor agonists exenatide 141758-74-9 liraglutide 204656-20-2 Alphaglucosidase inhibitors acarbose 56180-94-0 miglitol 72432-03-2 voglibose 83480-29-9 Dipeptidyl peptidase 4 inhibitors alogliptin 850649-62-6 berberine 2086-83-1; 633-65-8; 633-66-9 dutogliptin 852329-66-9 gemigliptin 911637-19-9 linagliptin 668270-12-0 saxagliptin 361442-04-8 sitagliptin 654671-78-0 vildagliptin 274901-16-5 Glinides mitiglinide 145375-43-5 nateglinide 105816-04-4 repaglinide 135062-02-1 Sulfonylureas acetohexamide 968-81-0 carbutamide 339-43-5 chlorpropamide 94-20-2 glibenclamide 10238-21-8 glibornuride 26944-48-9 glipizide 29094-61-9 glimepiride 93479-97-1 gliclazide 21187-98-4 gliquidone 33342-05-1 glisentide 32797-92-5 glyclopyramide 631-27-6 tolbutamide 64-77-7 tolazamide 1156-19-0 Fibrate bezafibrate 41859-67-0 ciprofibrate 52214-84-3 clofibrate 637-07-0; 882-09-7; 39087-48-4; 14613-30-0 fenofibrate 49562-28-9; 42017-89-0; 856676-23-8 gemfibrozil 25812-30-0 Thiazolidinediones rosiglitazone 122320-73-4; 302543-62-0; 155141-29-0; 397263-60-4 pioglitazone 111025-46-8; 112529-15-4 Biguanides buformin 1190-53-0 metformin 657-24-9; 1115-70-4 phenformin 834-28-6 Others bromocriptine 22260-51-1 chromium 14639-25-9 picolinate colesevelam 182815-44-7 dexfenfluramine 3239-44-9 imidapril 89396-94-1 inulin 9005-80-5 lipoic acid 62-46-4 mecobalamin 13422-55-4 Orlistat 96829-58-2 Insulin insulin 9004-10-8; 11070-73-8; 12584-58-6; 11061-68-0; 8063-29-4; 9004-21-1; 68859-20-1; 8049-62-5; 53027-39-7; 9004-17-5; 116094-23-6; 9004-12-0; 51798-72-2; 11091-62-6 169148-63-4; 160337-95-1; 207748-29-6; 133107-64-9; 874442-57-6

A more preferred object of this invention relates to the above mentioned compositions of the invention in combination with one compound selected from the group consisting of glibenclamide, repaglinide, metformin and pioglitazone, as well as to the use of such combinations in the treatment of diabetic neuropathies in subject in need thereof.

Another preferred object of this invention relates to compositions relates to the above mentioned compositions of the invention in combination with one compound selected from the group consisting of pioglitazone, rosiglitazone, bezafibrate, ciprofibrate, clofibrate, fenofibrate, gemfibrozil, buformin, colesevelam, orlistat, as well as to the use of such combinations in the treatment of diabetic neuropathies in subject in need thereof.

In a particularly preferred embodiment, the invention relates to the above mentioned compositions in combination with metformin as well as to the use of such combinations in the treatment of diabetic neuropathies in subject in need thereof.

Therapy according to the invention may be provided at home, the doctor's office, a clinic, a hospital's outpatient department, or a hospital, so that one can observe the therapy's effects closely and make any adjustments that are needed as a function of measured blood glucose level.

The duration of the therapy depends on the stage of the disease being treated, age and condition of the patient, and how the patient responds to the treatment. The dosage, frequency and mode of administration of drugs or each component of the drug combinations of the invention can be controlled independently. For example, one drug of a combination may be administered orally while the second drug may be administered intramuscularly or at different times through the day. The drugs may also be formulated together such that one administration delivers all drugs.

The treatment of the invention can be administered during particular periods of the day, for example, on time or just before or just after the time the glucose concentration reaches its peak in the plasma.

Subjects at risk of developing diabetic neuropathies are subjects experiencing a perturbation of glucose homeostasis, and more particularly people suffering from hyperglycaemia but also pre-diabetes as these complications are frequently present prior to overt hyperglycaemia and diabetes. Though being possibly unnoticed by the patient, such perturbation of glucose homeostasis can be easily detected by measure of glycaemia. Pre-diabetes is diagnosed when fasting blood glucose exceeds 5.6 mmol/L and/or 2-hour glucose in a glucose tolerance test exceeds 6.9 mM. Diabetes is diagnosed when fasting blood glucose exceeds 6.9 mmol/L, or casual or 2-hour glucose in a glucose tolerance test exceeds 11 mmol/L. Glycaemia can be easily determined, even by the patients themselves, using different commercially available glucometers. The time and dosage of the treatment can therefore be adapted as a function of the measured glycaemia.

While it is possible for the drug or the drugs of the combination to be administered as the pure chemical, it is preferable to present them as a pharmaceutical composition, also referred to in this context as pharmaceutical formulation. Possible compositions include those suitable for oral, rectal, topical (including transdermal, buccal and sublingual), or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.

More commonly these pharmaceutical formulations are prescribed to the patient in “patient packs” containing a number dosing units or other means for administration of metered unit doses for use during a distinct treatment period in a single package, usually a blister pack. Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in traditional prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions. Thus, the invention further includes a pharmaceutical formulation, as herein before described, in combination with packaging material suitable for said formulations. In such a patient pack the intended use of a formulation for the combination treatment can be inferred by instructions, facilities, provisions, adaptations and/or other means to help using the formulation most suitably for the treatment. Such measures make a patient pack specifically suitable for and adapted for use for treatment with the compositions of the present invention.

The drug(s) may be contained, in any appropriate amount, in any suitable carrier substance. The drug(s) may be present in an amount of up to 99% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for the oral, parenteral (e.g., intravenously, intramuscularly), rectal, cutaneous, nasal, vaginal, inhalant, skin (patch), or ocular administration route. Thus, the composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols.

The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy [20] and Encyclopedia of Pharmaceutical Technology [21]).

Pharmaceutical compositions according to the invention may be formulated to release the active drug substantially immediately upon administration or at any predetermined time or time period after administration.

The controlled release formulations include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain drug action during a predetermined time period by maintaining a relatively, constant, effective drug level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active drug(s) substance; (iv) formulations that localize drug action by, e.g., spatial placement of a controlled release composition adjacent to or in the diseased tissue or organ; and (v) formulations that target drug action by using carriers or chemical derivatives to deliver the drug(s) to a particular targeted cell type.

Administration of drugs in the form of a controlled release formulation is especially preferred in cases in which the drug has (i) a narrow therapeutic index (i.e., the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; in general, the therapeutic index, TI, is defined as the ratio of median lethal dose (LD50) to median effective dose (ED50)); (ii) a narrow absorption window in the gastrointestinal tract; or (iii) a very short biological half-life so that frequent dosing during a day is required in order to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the drug in question. Controlled release may be obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the drug is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the drug in a controlled manner (single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes).

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the composition of the invention in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., stearic acid, silicas, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active drug substance in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug substance until after passage of the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose). A time delay material such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, (e.g., chemical degradation prior to the release of the active drug substance). The coating may be applied on the solid dosage form in a similar manner as that described in Encyclopedia of Pharmaceutical Technology.

Drugs may be mixed together in the tablet, or may be partitioned. For example, a first drug is contained on the inside of the tablet, and a second drug is on the outside, such that a substantial portion of the second drug is released prior to the release of the first drug.

Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, liquid paraffin, or olive oil. Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner.

Controlled release compositions for oral use may, e.g., be constructed to release the active drug by controlling the dissolution and/or the diffusion of the active drug substance.

Dissolution or diffusion controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of drugs, or by incorporating the drug into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated metylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.

A controlled release composition containing one or more of the drugs of the claimed combinations may also be in the form of a buoyant tablet or capsule (i.e., a tablet or capsule that, upon oral administration, floats on top of the gastric content for a certain period of time). A buoyant tablet formulation of the drug(s) can be prepared by granulating a mixture of the drug(s) with excipients and 20-75% w/w of hydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, or hydroxypropylmethylcellulose. The obtained granules can then be compressed into tablets. On contact with the gastric juice, the tablet forms a substantially water-impermeable gel barrier around its surface. This gel barrier takes part in maintaining a density of less than one, thereby allowing the tablet to remain buoyant in the gastric juice.

Liquids for Oral Administration

Powders, dispersible powders, or granules suitable for preparation of an aqueous suspension by addition of water are convenient dosage forms for oral administration. Formulation as a suspension provides the active ingredient in a mixture with a dispersing or wetting agent, suspending agent, and one or more preservatives. Suitable suspending agents are, for example, sodium carboxymethylcellulose, methylcellulose, sodium alginate, and the like.

Parenteral Compositions

The pharmaceutical composition may also be administered parenterally by injection, infusion or implantation (intravenous, intramuscular, subcutaneous, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation.

Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active drug(s), the composition may include suitable parenterally acceptable carriers and/or excipients. The active drug(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. The composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, and/or dispersing agents.

The pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable active drug(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the drugs is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.

Controlled release parenteral compositions may be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions. Alternatively, the active drug(s) may be incorporated in biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices. Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactin, poly-(isobutyl cyanoacrylate) or poly-(2-hydroxyethyl-L-glutamnine). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be non-biodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(glycolic acid) or poly(ortho esters)).

Alternative Routes

Although less preferred and less convenient, other administration routes, and therefore other formulations, may be contemplated. In this regard, for rectal application, suitable dosage forms for a composition include suppositories (emulsion or suspension type), and rectal gelatin capsules (solutions or suspensions). In a typical suppository formulation, the active drug(s) are combined with an appropriate pharmaceutically acceptable suppository base such as cocoa butter, esterified fatty acids, glycerinated gelatin, and various water-soluble or dispersible bases like polyethylene glycols. Various additives, enhancers, or surfactants may be incorporated.

The pharmaceutical compositions may also be administered topically on the skin for percutaneous absorption in dosage forms or formulations containing conventionally non-toxic pharmaceutical acceptable carriers and excipients including microspheres and liposomes. The formulations include creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters, and other kinds of transdermal drug delivery systems. The pharmaceutically acceptable carriers or excipients may include emulsifying agents, antioxidants, buffering agents, preservatives, humectants, penetration enhancers, chelating agents, gel-forming agents, ointment bases, perfumes, and skin protective agents.

The preservatives, humectants, penetration enhancers may be parabens, such as methyl or propyl p-hydroxybenzoate, and benzalkonium chloride, glycerin, propylene glycol, urea, etc.

The pharmaceutical compositions described above for topical administration on the skin may also be used in connection with topical administration onto or close to the part of the body that is to be treated. The compositions may be adapted for direct application or for application by means of special drug delivery devices such as dressings or alternatively plasters, pads, sponges, strips, or other forms of suitable flexible material.

Dosages and Duration of the Treatment

Composition according to the invention is administered to a subject orally or by subcutaneous, intravenous or intramuscular injections, at different times of day, depending for example of blood glucose level. Nevertheless glucose level being not necessarily the only factor underlying pathogenesis of diabetic neuropathies, a long term and chronic treatment of patient known to be at risk for such diseases is desirable.

It will be appreciated that the drugs of a combination may be administered concomitantly, either in the same or different pharmaceutical formulation or sequentially. A minimum requirement for a combination according to this description is that the combination should be intended for combined use with the benefit of the efficacious effect of the combination of the active ingredients. The intended use of a combination can be inferred by facilities, provisions, adaptations and/or other means to help using the combination according to the invention.

Therapeutically effective amounts of the drugs in a combination of this invention include, e.g., amounts that are effective improving diabetic neuropathies.

Administration can be one to several times daily for several days to several years, and may even be for the life of the patient. Chronic or at least periodically repeated long-term administration is indicated in most cases.

The term “unit dosage form” refers to physically discrete units (such as capsules, tablets, or loaded syringe cylinders) suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material or materials calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier.

The amount of each drug in a preferred unit dosage composition depends upon several factors including the administration method, the body weight and the age of the patient, the stage of the disease, the risk of potential side effects considering the general health status of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage used.

Except when responding to especially impaired glucose levels where higher dosages may be required, the preferred dosage of each drug in the combination will usually lie within the range of doses not above the dosage usually prescribed for long-term maintenance treatment or proven to be safe in phase 3 clinical studies.

One remarkable advantage of the invention is that each compound may be used at low doses in a combination therapy, while producing, in combination, a substantial clinical benefit to the patient. The combination therapy may indeed be effective at doses where the compounds have individually low or no effect. Accordingly, a particular advantage of the invention lies in the ability to use sub-optimal doses of each compound, i.e., doses which are lower than therapeutic doses usually prescribed, preferably ½ of therapeutic doses, more preferably ⅓, ¼, ⅕, or even more preferably 1/10 of therapeutic doses. In particular examples, doses as low as 1/20, 1/30, 1/50, 1/100, or even lower, of therapeutic doses are used.

Such a clinical benefit at low doses includes synergy. Synergy can be proven through different ways, for instance, by calculating a combinatory index from dose-effect curves of each of the compounds alone and of their combinations and/or using the factorial ANOVA test with treatments as factors, indicating whether an interaction between the factors is significant. Synergy may be assessed by methods known by those skilled in the art. At sub-therapeutic dosages, the compounds would exhibit no or less side effects, while the combinations according to the invention are fully effective in treating diabetic neuropathies, promoting nerve regeneration in a subject suffering from, at risk of suffering from, or likely to suffer from a diabetic neuropathy, or protecting cells against the stresses resulting from the perturbation of glucose homeostasis.

A preferred dosage corresponds to amounts from 1% up to 50% of those usually prescribed for long-term maintenance treatment.

The most preferred dosage may correspond to amounts from 1% up to 10% of those usually prescribed for long-term maintenance treatment.

Specific examples of dosages of drugs for use in the invention are provided below:

-   -   acamprosate from about 9 to 200 mg per day, preferably orally,     -   almitrine from about 0.5 to 10 mg per day, preferably orally,     -   amlexanox from about 0.75 to 15 mg per day, preferably orally,     -   baclofen from about 0.15 to 50 mg per day, preferably orally,     -   carbetapentane from about 0.6 to 18 mg per day, preferably         orally,     -   cimetidine from about 4 to 160 mg per day, preferably orally,     -   cinacalcet from about 0.3 to 36 mg per day, preferably orally,     -   dexbrompheniramine from about 0.06 to 1.2 mg per day, preferably         orally,     -   diethylcarbamazine from about 0.6 to 600 mg per day, preferably         orally,     -   diprophylline from about 9 to 320 mg per day, preferably orally,     -   fenspiride from about 1.6 to 24 mg per day, preferably orally,     -   fexofenadine from about 1.2 to 18 mg per day, preferably orally,     -   idebenone from about 4.5 mg to 225 mg per day, preferably         orally,     -   ifenprodil from about 0.4 to 6 mg per day, preferably orally,     -   mexiletine from about 6 to 120 mg per day, preferably orally,     -   nicergoline from about 0.6 to 6 mg per day, preferably orally,     -   piribedil from about 0.8 to 25 mg per day, preferably orally,     -   rilmenidine from about 10 to 200 μg per day, preferably orally,     -   tolfenamic acid from about 3 to 60 mg per day, preferably         orally,     -   tolperisone from about 1.5 to 4.5 mg per day, preferably orally,     -   torasemide from about 0.05 to 4 mg per day, preferably orally,     -   triamterene from about 1.5 to 25 mg per day, preferably orally.

In combinations of the invention, the molar ratio between drugs may vary e.g., from 0.001 to 1000. Also, the ratio of the drug(s) and excipient in a composition of the invention advantageously vary between 0.001 and 1000.

It will be understood that the amount of the drug(s) actually administered will be determined by a physician, in the light of the relevant circumstances including the condition or conditions to be treated, the exact composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration. Therefore, the above dosage ranges are intended to provide general guidance and support for the teachings herein, but are not intended to limit the scope of the invention.

The following examples are given for purposes of illustration and not by way of limitation.

Examples

Diabetic neuropathy is notably the result of high blood glucose levels occurring in diabetes. Hence, the efficacy of candidate compounds is determined based on several in vitro and in vivo studies in order to address most of the metabolic and physiological impairments characterizing this complex pathology. The properties of the compounds are determined using models which illustrate physiological features representative of a neuropathy induced by an abnormal blood glucose level.

I. Compounds of the Invention are Efficient in Protecting Neurite Outgrowth In Vitro.

a) Neurite Network from Rat Cortical Cells

Culture Conditions

Rat cortical neurons are cultured as described by Singer et al. [22]. Briefly, pregnant female rats of 15 days gestation are killed by cervical dislocation (rats Wistar) and the foetuses are removed from the uterus. The cortex is then removed and placed in ice-cold medium of Leibovitz (L15) containing 2% of Penicillin 10.000 U/mL and Streptomycin 10 mg/mL and 1% of bovine serum albumin (BSA). Cortices are dissociated by trypsin for 20 min at 37° C. (0.05%). The reaction is stopped by the addition of Dulbecco's modified Eagle's medium (DMEM) containing DNasel grade II and 10% of foetal calf serum (FCS). Cells are then mechanically dissociated by 3 serial passages through a 10 mL pipette and centrifuged at 515×g for 10 min at +4° C. The supernatant is discarded and the pellet of cells is re-suspended in a defined culture medium consisting of Neurobasal supplemented with B27 (2%), L-glutamine (0.2 mM), 2% of PS solution and 10 ng/mL of BDNF. Viable cells are counted in a Neubauer cytometer using the trypan blue exclusion test. The cells are seeded at a density of 30 000 cells/well in 96 well-plates (wells were pre-coated with poly-L-lysine (10 μg/mL)) and were cultured at +37° C. in a humidified air (95%)/CO2 (5%) atmosphere.

After 10 days of culture, cells are incubated with drugs and their combinations. After 1 hour, medium glucose concentration is raised to 45 mmol/L glucose in defined medium without BDNF but together with tested compound. Cortical neurons are so cultured for 24 hours. BDNF (10 ng/mL) is used as a positive (neuroprotective) control. Three independent cultures were performed per condition, 6 wells per condition.

Neurites Length Quantitation

After 24 hours of culture in high glucose conditions, the supernatant is taken off and the cortical neurons are fixed by a cold solution of ethanol (95%) and acetic acid (5%) for 5 min. After permeabilization with 0.1% of saponin, cells are blocked for 2 hours with PBS containing 1% foetal calf serum. Then, cells are incubated with monoclonal antibody anti microtubule-associated-protein 2 (MAP-2; Sigma). This antibody stains specifically cell bodies and neurites of neurons. Anti-Map2 antibody is revealed with Alexa Fluor 488 goat anti-mouse IgG (Molecular probe). Nuclei of neurons are labelled by a fluorescent marker (Hoechst solution, Sigma).

Per well, 10 pictures are taken using InCell Analyzer™ 1000 (GE Healthcare) with 20× magnification. All pictures are taken in the same conditions. Analysis of the neurite network is done using Developer software (GE Healthcare) in order to assess the total length of neurite network.

Results

As mentioned in table 4, drugs and drug combinations identified by the inventors induce a significant improvement in neurite network length of primary cortical neuron cell cultures which is otherwise significantly reduced under high glucose conditions.

TABLE 4 Protection of neurite network drugs/ rat cortical primary DRG explants drug combinations cell culture from STZ rats acamprosate + + baclofen + + carbetapentane + + cimetidine + + cinacalcet + + dexbrompheniramine + + diethylcarbamazine + + ifenprodil + + levosimendan + + mexiletine + + nicergoline + + rilmenidine + + tolperisone + + torasemide + + triamterene + + acamprosate and cinacalcet + + acamprosate and levosimendan + + acamprosate and torasemide, + + acamprosate and triamterene, + + acamprosate and baclofen, + + cinacalcet and levosimendan, + + cinacalcet and torasemide, + + cinacalcet and triamterene, + + torasemide and triamterene, + + levosimendan and torasemide, + + levosimendan and triamterene + + +: significant improvement in neurite network length when compared to non-treated cultures.

b) Protective Effect Toward Neurite Outgrowth from Dorsal Root Ganglia Explants from STZ Rats

Experimental Diabetes

Diabetes is induced in Wistar rats (300-320 g) by intraperitoneal injection of streptozotocin (50 mg/kg; STZ). Diabetes is confirmed in all STZ-treated rats 2 days later by measuring glucose concentration of tail-vein blood. Normal and diabetic rats are maintained on a 12 hours light/dark cycle with free access to food and water for 12 weeks.

Primary Dorsal Root Ganglia Cultures and Neurite Outgrowth

Whole transverse slices of Wistar rats (Janvier Labs) spinal cords with dorsal root ganglia (DRG) attached are maintained in a mixed (75%/25%) medium composed of MEM and medium 199, supplemented with 5% FCS, insulin 5 μg/mL, glutamine 2 mM and 2% PS. After 24 hours of culture, neurites are observed growing out of the spinal cord explants.

After incubation with drugs and drug combinations, cells are incubated with 500 nM α-bungarotoxin coupled with Alexa 488 during 15 min in culture innervations medium at 37° C. After 2 washings in PBS, cells are fixed by a solution of 4% of paraformaldehyde in PBS, pH of 7.3, for 20 min at room temperature.

The cells are washed 2 times in PBS and then permeabilized and non-specific sites are blocked with a solution of PBS containing 0.1% of saponin and 1% FCS for 15 min at room temperature. Cultures are incubated with a mouse monoclonal anti-neurofilament 200 KD antibody (NF) at the dilution of 1/400 in PBS containing 1% FCS, 0.1% saponin, for 2 hours at room temperature. Antibody against NF stained the axon of neurons. These antibodies are revealed with Alexa Fluor 568 goat anti-mouse IgG at the dilution 1/400 in PBS containing 1% FCS, 0.1% saponin, for 1 hour at room temperature. Nuclei of neurons are labelled by Hoechst solution, a nuclear fluorescent marker at 1 μm/mL in the same solution.

Three independent cultures are done. For each condition, 20 pictures per well are taken using InCell Analyzer™ 1000 (GE Healthcare) with 10× magnification. All the images are taken under the same conditions. Total neurite length is quantified.

Treatment

Spinal cord explants harvested from normal or diabetic rats are cultured 24-48-72 hours without or with tested compounds. Duloxetine or pregabalin treatment is used a positive control.

Results

Measured length of neurite network is significantly lower for culture from DRG explants from STZ treated rats than in non-diabetic rats. As mentioned in table 4, compounds and their combinations identified by the inventors significantly promote growth of neurites from DRG explants of diabetic rats when cultured with compounds of the invention (table 4).

c) Co-Culture Model of Sensory Nerves and Schwann Cells

A model of co-culture was used as an in vitro model of diabetes. This model of myelination consists in co-culturing sensory neurons and Schwann cells from Wistar rats dissociated dorsal root ganglia (DRG).

The aim of this study is to assess the effect of tested compounds and compound combinations on myelination process under high glucose conditions mimicking diabetes. The effect of the tested compounds on myelination, either in high glucose concentration or not, is assessed by evaluating Myelin Basic Protein (MBP) expression in presence of ascorbic acid.

Sensory Neurons and Schwann Cells Co-Cultures

Fifteen days gestation pregnant female rats are killed by cervical dislocation. The embryos are removed from the uterus and are at similar foetal stage of development.

Rat DRG are cultured as previously described by Cosgaya et al. [23] and Rangaraju et al. [24].

Each embryo is dispatched on numerating petri dish (35 mm of diameter). DRG of each embryo is collected, placed in ice-cold medium of Leibovitz (L15, Invitrogen). At the end of the dissection, DRG of TGM are pooled and dissociated by trypsinization (trypsin EDTA, 0.05%; Invitrogen) for 20 min at 37° C. The reaction is stopped by addition of DMEM containing 10% of foetal bovine serum (FBS) in the presence of DNAase I (Roche). The suspension is triturated with a 10 mL pipette. Cells are then centrifuged at 350×g for 10 min at room temperature. The pellet of dissociated cells is resuspended in neurobasal medium (Invitrogen) containing 2% of B27 (Invitrogen), 1% of penicillin-streptomycin (Invitrogen), 1% de L glutamine and 50 ng/mL NGF (Sigma). This medium is the neuronal medium. Viable cells are counted in a Neubauer cytometer using the trypan blue exclusion test (Sigma) and seeded on the basis of 10 000 cells per well in 96 well-plates (Greiner) treated with poly-L-lysine. The plates are maintained at 37° C. in a humidified incubator, in an atmosphere of air (95%)-CO2 (5%). Half of the standard neuronal culture medium is changed every other day. The cultures are maintained in standard neurobasal medium for 7 days to allow Schwann cells to populate the sensory neuron neurites. On day 7, the cultures are fed with standard neuronal medium supplemented or not with 50 μg/mL ascorbic acid in order to initiate basal lamina formation and myelination.

Drug Incubation-Diabetes Mimicking Conditions

On Day 7, the Following Tested Compounds (Alone or in Combination) Listed in Table 5 are added in the medium with 50 μg/mL ascorbic acid. Basal glucose in culture medium is 25 mmol/L, which is necessary for DRG neuron survival. To produce a hyperglycemic insult, 20 mmol/L additional glucose (total 45 mmol/L glucose) is added to the media for the period specified.

The tested compounds are incubated for 5 different times: 5, 9, 10, 11 and 13 days.

Three separate and independent cultures of DRG are done. These conditions are assessed in presence of ascorbic acid, 6 wells per condition. The solution ready to use of all tested compounds are extemporaneously prepared from a stock solution, stored at −20° C. This solution is prepared once a week. Half of the standard neuronal medium supplemented with tested compounds, ascorbic acid (each at the concentration 1×) and glucose are changed every other day.

Staining Protocol

After 5, 9, 10, 11 and 13 days of incubation, cells are fixed by a cold solution of ethanol (95%) and acetic acid (5%) for 10 min. The cells are permeabilized and blocked with PBS containing 0.1% saponin and 10% goat serum for 15 min. Then, the cells are incubated with a specific marker of myelin: polyclonal antibody anti-myelin basic protein (MBP) antibody (Sigma 118K0431).

This antibody is revealed with Alexa Fluor 568 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG (Molecular probe 687621, 623962). Nuclei of neurons are labelled by a fluorescent marker (Hoechst solution, SIGMA ref B1155).

Data Processing

Per well, 20 pictures are taken using InCell Analyzer™ 1000 (GE Healthcare) with 20× magnification. All images are taken in the same conditions. Analysis of total length of myelinated axons was automatically done (length and area around axons) using

Developer software (GE Healthcare). All values will be expressed as mean+/−s.e.mean. Statistical analyses are done on different conditions (ANOVA followed by Fisher's PLSD test when allowed).

Results

A significant effect on myelination is observed upon incubation with all the tested compounds of the invention whereas hyperglycemic conditions induce an important drop in myelination of non-treated co-cultures.

TABLE 5 Compound Myelinated axon length acamprosate + baclofen + carbetapentane + cimetidine + cinacalcet + dexbrompheniramine + diethylcarbamazine + ifenprodil + levosimendan + mexiletine + nicergoline + rilmenidine + tolperisone + torasemide + triamterene + acamprosate and cinacalcet + acamprosate and levosimendan + acamprosate and torasemide, + acamprosate and triamterene, + Acamprosate and baclofen + cinacalcet and levosimendan, + cinacalcet and torasemide, + cinacalcet and triamterene, + torasemide and triamterene, + levosimendan and torasemide, + levosimendan and triamterene + +: significant improvement in length of myelinated axons when compared to non-treated conditions as assessed by MBP expression.

II. Compounds of the Invention are Efficient in Lessening Diabetic Neuropathy Symptoms In Vivo.

Animals and Induction of Diabetes

Male Sprague-Dawley rats weighing 200 to 250 g are used. They are housed three per cage under standard laboratory conditions, and given food and water ad libitum.

The rats are rendered diabetic with an intra-peritoneal injection of STZ (75 mg/kg) dissolved in distilled water. Diabetes is confirmed 4 weeks later by measurement of tail vein blood glucose levels with Ames Dextrostix and a reflectance colorimeter. Only rats with a final blood glucose level ≧14 mM are included in the study. Control (normal) rats receive only distilled water.

Treatment

Tests take place 4 weeks after the induction of diabetes. Only rats in which the reduction in pain scores at week 4 of diabetes is more than 15% of the value obtained in normal rats are included. The animals are submitted to the nociceptive test (paw pressure) before treatment.

Once two stable threshold values are obtained, treatment made of vehicle with tested compound, or with duloxetine or pregabalin (30 mg/kg/day, per os) as positive control, or of vehicle alone, is administered. Rats are arranged randomly in cages, each rat receiving treatment in the same volume (0.1 mL/100 g b.wt.).

The experiments are performed blind by the method of equal blocks with randomization of treatments (n=7 for each treatment). Different animals are used for each test and treatment.

Behavioral Studies

Mechanical Stimulus: The Paw Pressure Test.

Nociceptive thresholds, expressed in grams, are measured with a Ugo Basil analgesimeter (tip diameter of probe, 1 mm; weight, 30 g) by applying an increasing pressure to the left hind paw until withdrawal (cut-off was 750 g).

Mechanical Hyperalgesia: Electronic Von Frey Test.

Mechanical hyperalgesia is assessed by measuring the withdrawal threshold of the left hind paw in response to a mechanical stimulus using an electronic von Frey aesthesiometer. A rigid tip attached to the meter is applied to the left plantar surface from under the floor. The withdrawal threshold is defined as the average force (g) required causing withdrawal of the stimulated paw in three trials.

Cold Allodynia: Acetone Test.

Cold allodynia is assessed using the acetone test. In this test, latency of hind-paw withdrawal is measured after application of a drop of acetone to the plantar surface of both hind-paws (reaction time) and the intensity of the response is scored (cold score). Reaction time to the cooling effect of acetone is measured within 20 sec (cut-off) after acetone application. Responses to acetone are also graded to the following 4-point scale: 0 (no response); 1 (quick withdrawal, flick of the paw); 2 (prolonged withdrawal or marked flicking of the paw); 3 (repeated flicking of the paw with licking or biting).

Data Processing

Statistical significance between the treated groups and the vehicle-treated group is determined by a two-way repeated measures ANOVA followed, when the F value is significant, by an appropriate post-hoc comparison (Bonferroni's test). The significance level is set at p<0.05.

Results

All the tested combinations of compounds of the invention are found efficient in lowering the intensity of at least one of neuropathic symptoms measured in STZ treated rats (table 6).

TABLE 6 Mechanical stimulus and/or mechanical Combination of compounds hyperalgesia and/or cold allodynia acamprosate + cinacalcet + acamprosate + levosimendan + acamprosate + torasemide + acamprosate + triamterene + acamprosate + baclofen + cinacalcet + levosimendan + cinacalcet + torasemide + cinacalcet + triamterene + torasemide + triamterene + levosimendan + torasemide + levosimendan + triamterene + + means an improvement of analgesia, hyperalgesia, and/or cold allodynia symptoms observed in diabetic rats (STZ induced diabetes).

Hence as shown above, compounds of the invention are efficient in treating in vitro or in vivo diabetic neuropathies or symptoms thereof, through re-myelination processes, axonal growth, enhancement of neuronal growth, and/or restoration of either morphological or functional features of the nerve. Such compounds represent therefore valuable treatments for diabetic neuropathies.

BIBLIOGRAPHY

-   1 Brownlee M (2001) Biochemistry and molecular cell biology of     diabetic complications. Nature 414, 813-20. -   2 Vincent A M, McLean L L, Backus C & Feldman E L (2005) Short-term     hyperglycemia produces oxidative damage and apoptosis in neurons.     FASEB J. 19, 638-40. -   3 Goldstein D J, Lu Y, Detke M J, Lee T C & Iyengar S (2005)     Duloxetine vs. placebo in patients with painful diabetic neuropathy.     Pain 116, 109-18. -   4 Bril V, England J, Franklin G M, Backonja M, Cohen J, Del Toro D,     Feldman E, Iverson D J, Perkins B, Russell J W & Zochodne D (2011)     Evidence-based guideline: Treatment of painful diabetic neuropathy:     report of the American Academy of Neurology, the American     Association of Neuromuscular and Electrodiagnostic Medicine, and the     American Academy of Physical Medicine and Rehabilitation. Neurology     76, 1758-65. -   5 Jensen T S, Backonja M-M, Hernández Jiménez S, Tesfaye S, Valensi     P & Ziegler D (2006) New perspectives on the management of diabetic     peripheral neuropathic pain. Diab. Vasc. Dis. Res. 3, 108-19. -   6 Ziegler D, Ametov A, Barinov A, Dyck P J, Gurieva I, Low P A,     Munzel U, Yakhno N, Raz I, Novosadova M, Maus J & Samigullin     R (2006) Oral treatment with alpha-lipoic acid improves symptomatic     diabetic polyneuropathy: the SYDNEY 2 trial. Diabetes Care 29,     2365-70. -   7 Stracke H, Gaus W, Achenbach U, Federlin K & Bretzel R G (2008)     Benfotiamine in diabetic polyneuropathy (BENDIP): results of a     randomised, double blind, placebo-controlled clinical study. Exp.     Clin. Endocrinol. Diabetes 116, 600-5. -   8 Ziegler D, Movsesyan L, Mankovsky B, Gurieva I, Abylaiuly Z &     Strokov I (2009) Treatment of symptomatic polyneuropathy with     actovegin in type 2 diabetic patients. Diabetes Care 32, 1479-84. -   9 Leach A R & Gillet V J An Introduction to Chemoinformatics     (Springer-Verlag New York Inc, ed.). -   10 Rahman S A, Bashton M, Holliday G L, Schrader R & Thornton J     M (2009) Small Molecule Subgraph Detector (SMSD) toolkit. J.     Cheminform. 1, 12. -   11 Stella V J (2007) Prodrugs: challenges and rewards. (A. Press and     Springer, eds.) Springer Singapore Pte. Limited, New-York. -   12 Wermuth C G (2011) The Practice of Medicinal Chemistry Elsevier     Science. -   13 Pezron I, Mitra A K, Duvvuri S & Tirucherai G S (2002) Prodrug     strategies in nasal drug delivery. Expert Opin. Ther. Pat. 12,     331-340. -   14 Stella V J (2004) Prodrugs as therapeutics. Expert Opin. Ther.     Pat. 14, 277-280. -   15 Stella V J & Nti-Addae K W (2007) Prodrug strategies to overcome     poor water solubility. Adv. Drug Deliv. Rev. 59, 677-94. -   16 Beaumont K, Webster R, Gardner I & Dack K (2003) Design of ester     prodrugs to enhance oral absorption of poorly permeable compounds:     challenges to the discovery scientist. Curr. Drug Metab. 4, 461-85. -   17 Higuchi T & Stella V J (1975) Pro-drugs as Novel Drug Delivery     System, ACS Sympos American Chemical Society, Washington, D.C. -   18 Roche E B (1977) Design of biopharmaceutical properties through     prodrugs and analogs: a symposium, American P The Academy,     Washington, D.C. -   19 Stahl H & Wermuth C G (2011) Pharmaceutical salts: Properties,     selection, and use, 2nd ed. (Wiley-VCH, ed.). -   20 Gennaro A R (2000) Remington: The Science and Practice of     Pharmacy, 20th ed. (A. D. Gennaro, W. Lippincott, and Wilkins, eds.)     Lippincott Williams & Wilkins -   21 Swarbrick J & Boylan J C (eds.) Encyclopedia of Pharmaceutical     Technology Dekker, Marcel, New-York. -   22 Singer C A, Figueroa-Masot X A, Batchelor R H & Dorsa D M (1999)     The mitogen-activated protein kinase pathway mediates estrogen     neuroprotection after glutamate toxicity in primary cortical     neurons. J. Neurosci. 19, 2455-63. -   23 Cosgaya J M, Chan J R & Shooter E M (2002) The neurotrophin     receptor p75NTR as a positive modulator of myelination. Science 298,     1245-8. -   24 Rangaraju S, Madorsky I, Pileggi J G, Kamal A & Notterpek     L (2008) Pharmacological induction of the heat shock response     improves myelination in a neuropathic model. Neurobiol. Dis. 32,     105-15. 

1. A method for the treatment of a diabetic neuropathy in a subject in need thereof comprising administering to the subject a composition comprising at least two drugs selected from acamprosate, almitrine, amlexanox, azelastine, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or salts, prodrugs, derivatives, or sustained release formulations thereof.
 2. The method of claim 1, comprising administering to the subject: at least one drug selected from acamprosate, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, ifenprodil, levosimendan, mexiletine, nicergoline, rilmenidine, tolperisone, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof, and at least one distinct drug selected form acamprosate, almitrine, amlexanox, azelastine, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof.
 3. The method of claim 1, comprising administering to the subject: at least one drug selected from acamprosate, cinacalcet, levosimendan, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof, and at least one distinct drug selected form acamprosate, almitrine, amlexanox, azelastine, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, diprophylline, D-mannose, fenspiride, fexofenadine, idebenone, ifenprodil, levosimendan, mexiletine, nicergoline, piribedil, rilmenidine, tolfenamic acid, tolperisone, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof.
 4. The method of claim 1, comprising administering to the subject: at least one drug selected from acamprosate, cinacalcet, levosimendan, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof, and at least one distinct drug selected form acamprosate, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, ifenprodil, levosimendan, mexiletine, nicergoline, rilmenidine, tolperisone, torasemide and triamterene, or a salt, prodrug, derivative, or sustained release formulation thereof.
 5. The method of claim 1, comprising administering to the subject at least two drugs selected from acamprosate, baclofen, carbetapentane, cimetidine, cinacalcet, dexbrompheniramine, diethylcarbamazine, ifenprodil, levosimendan, mexiletine, nicergoline, rilmenidine, tolperisone, torasemide and triamterene, or salts, prodrugs, derivatives, or sustained release formulations thereof.
 6. The method of claim 1, comprising administering to the subject at least one of the following drug combinations: acamprosate and cinacalcet, acamprosate and levosimendan, acamprosate and torasemide, acamprosate and triamterene, acamprosate and baclofen, cinacalcet and levosimendan, cinacalcet and torasemide, cinacalcet and triamterene, torasemide and triamterene, levosimendan and torasemide, or levosimendan and triamterene, or salts, prodrugs, derivatives, or sustained release formulations thereof.
 7. The method of claim 1, further comprising administering to the subject at least one drug selected from duloxetine and pregabalin, or a salt, prodrug, derivative, or sustained release formulation thereof.
 8. The method of claim 1, further comprising administering to the subject at least one antidiabetic agent selected from acarbose, acetohexamide, alogliptin, berberine, bezafibrate, bromocriptine, buformin, carbutamide, chlorpropamide, chromium picolinate, ciprofibrate, clofibrate, colesevelam, dexfenfluramine, dutogliptin, exenatide, fenofibrate, gemfibrozil, gemigliptin, glibenclamide, glibornuride, glicetanile, gliclazide, glimepiride, glipizide, gliquidone, glisentide, glyclopyramide, imidapril, insulin, inulin, lipoic acid, linagliptin, liraglutide, mecobalamin, metformin, miglitol, mitiglinide, nateglinide, orlistat, phenformin, pioglitazone, pramlintide, repaglinide, rosiglitazone, saxagliptin, sitagliptin, tolazamide, tolbutamide, vildagliptin or voglibose, or a salt, prodrug, derivative, or sustained release formulation thereof.
 9. The method of claim 8, wherein the at least one further antidiabetic agent is metformin, or a salt, prodrug, derivative, or sustained release formulation thereof.
 10. The method of claim 1, wherein the subject is suffering from or at risk of suffering from a diabetic neuropathy.
 11. The method of claim 10, wherein the diabetic neuropathy is selected from a diabetic peripheral neuropathy, a diabetic proximal neuropathy, a diabetic autonomic neuropathy and a diabetic focal neuropathy.
 12. The method of claim 1, wherein the subject is suffering from diabetes type I or II, chronic hyperglycaemia, impaired fasting glycaemia, impaired glucose tolerance or obesity.
 13. A method of claim 1, for use in promoting nerve regeneration in a subject suffering from or at risk of suffering from a diabetic neuropathy.
 14. A method of claim 1, for use in protecting nervous system cells against the stresses resulting from the perturbation of glucose homeostasis.
 15. The method of claim 1, which wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.
 16. The method of claim 1, wherein the drugs in said composition are formulated or administered simultaneously, separately or sequentially.
 17. The method of claim 1, wherein said composition is administered repeatedly to the subject. 